Methods of Modifying Agricultural Co-Products and Products Made Therefrom

ABSTRACT

In a method of producing a polymer composite, a polymer is provided in a liquid state such as a molten state. A plant material, such as soymeal, is provided that includes protein and carbohydrate. The plant material has a particle size less than 50 microns. A reactive protein denaturant is also provided. A dispersion of the plant material and the reactive protein denaturant is formed in a matrix of the liquid polymer. The plant material is reacted to bond with the reactive protein denaturant, and the reactive protein denaturant is reacted to bond with the polymer. The polymer is solidified to produce the polymer composite.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part patent application of U.S.patent application Ser. No. 13/386,150 filed Apr. 24, 2012, which is theNational Stage of International Application PCT/US 10/42567 filed Jul.20, 2010, which claims the benefit of U.S. Provisional Application No.61/226,904 filed Jul. 20, 2009, and claims the benefit of No. 61/227,998filed Jul. 23, 2009, and claims the benefit of No. 61/245,695 filed Sep.25, 2009, the disclosures of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

This invention relates in general to plant materials and methods, and inparticular to methods of modifying agricultural co-products, andproducts such as polymer composites made with the modified co-products.

The high cost of petroleum-based fuels is resulting in increasedproduction of biofuels such as biodiesel and ethanol. This is expectedto result in an oversupply of agricultural co-products, which are theplant materials remaining after the plants are used to produce thebiofuel. For example, the production of biodiesel uses the oil fromsoybeans or other plants and leaves co-products such as flakes, stemsand hulls. Finding industrial applications for the co-products wouldimprove the economics of the biofuel industry and enhance theprofitability of farmers.

Oil extraction from grains generally results in co-products that containboth proteins and carbohydrates. The current practice is to isolate andpurify to separate the proteins and carbohydrates from each other forlater use as surfactants, rheology modifiers, fillers, etc. However,isolating proteins and carbohydrates from agricultural co-products is avery expensive process, which has hindered commercialization of theproducts.

The increase in oil prices has escalated the cost of petroleum-derivedpolymers. If the petroleum-derived polymers can be partially replaced byother materials, this substitution would save costs and reduceenvironmental waste associated with the polymers. Composites have beenproduced in which a portion of the polymer is replaced by plant-derivedmaterials, but the existing commercial products are either based onisolated proteins or carbohydrates as fillers.

It would be desirable to provide improved methods and products usingagricultural co-products and other plant materials.

SUMMARY OF THE INVENTION

This invention relates to a method of producing a polymer composite. Apolymer is provided in a liquid state such as a molten state. A plantmaterial, such as soymeal, is provided that includes protein andcarbohydrate. The plant material has a particle size less than 50microns. A reactive protein denaturant is provided. A dispersion of theplant material and the reactive protein denaturant is formed in a matrixof the liquid polymer. The plant material is reacted to bond with thereactive protein denaturant, and the reactive protein denaturant isreacted to bond with the polymer. The polymer is solidified to producethe polymer composite.

In a reactive extrusion method of producing the polymer composite, thedispersion is formed and the reactions are conducted in a single-stepprocess in an extruder.

Polymer composites produced by the methods are also described.

Various aspects of this invention will become apparent to those skilledin the art from the following detailed description of the preferredembodiment.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The methods and products of the invention can utilize any suitable plantmaterial that includes both protein and carbohydrate. Some nonlimitingexamples of plants that could be used include soybeans, corn, wheat,sesame, cotton, coconut, groundnut, palm, sugarcane, beets, sunflower,castor, grasses and weeds.

In certain embodiments, the plant material is an agriculturalco-product, which is plant material remaining after the plant has beenused to produce another product. For example, the production ofbiodiesel uses oil extracted from soybeans or other plants. To extractoil from soybeans, the soybeans are cracked, adjusted for moisturecontent, rolled into flakes and solvent-extracted. The remaining flakescan be comminuted to produce soybean meal, soybean flour or soybeangrit.

In certain embodiments, the particle size of the plant material iscontrolled. For example, the plant material may have a particle sizeless than 50 microns, more particularly less than 40 microns, moreparticularly less than 30 microns, and most particularly less than 20microns. It was surprisingly found that when the composites are formedfrom plant material having a smaller particle size, the resulting finalproduct has less odor and less residual color (e.g., browning). By“particle size” is meant the largest diameter of a particle. Theparticle size can be measured by any suitable method; for example, byU.S.A. Standard Sieves ASTM Specification E-11, other types of sieves orscreens, laser difraction, dynamic light scattering, or image analysis(dynamic or static). The particle size can be controlled by any suitableprocess; for example, by increased milling of the plant material.

In certain embodiments, the protein content of the plant material iscontrolled. For example, the plant material may have a protein contentless than 45%, more particularly less than 40%, and most particularlyless than 35%. It was surprisingly found that when the composites areformed from plant material having a lower protein content, the resultingfinal product has less odor and less residual color. The protein contentof the plant material can be measured by any suitable method; forexample, by the standard AOAC method.

This invention provides methods of modifying agricultural co-productsand other plant materials to make them more suitable for use inproducing commercial products and/or for use in other industrialapplications. In one embodiment, a method of modifying a plant materialcomprises the steps of: providing a plant material that includes proteinand carbohydrate; providing a reactive protein denaturant that iscapable of chemically bonding to the protein and promoting unfolding ofthe protein; and reacting to chemically bond the reactive proteindenaturant to the plant material under conditions sufficient to unfoldthe protein.

The reactive protein denaturant can be any material that is capable ofchemically bonding to the protein and promoting unfolding of theprotein. In certain embodiments, the reactive protein denaturant is anon-polymeric material. For example, in some particular embodiments, thereactive protein denaturant is a material selected from ethylenicallyunsaturated anhydrides, ethylenically unsaturated carboxylic acids,ethylenically unsaturated carboxylic acid esters, ethylenicallyunsaturated amines and imines, ethylenically unsaturated diketonates,derivatives of these materials, and mixtures of these materials. Someexamples of denaturants include maleic anhydride, acetoacetoxyethylmethacrylate, methacrylic acid, methyl methacrylate, butyl acrylate, andsoy acrylate.

In certain embodiments, the reactive protein denaturant is the productof the reaction between an unsaturated anhydride and an amino alcoholfunctionality. Some nonlimiting examples of such denaturants aredescribed in Sample B and Sample C of Example 9 hereinbelow. Examples ofan unsaturated anhydride include but are not limited to maleicanhydride, 1-cyclopentene-1,2-dicarboxylic anhydride,4,7-dihydro-2-benzofuran-1,3-dione, 2,3-dichloromaleic anhydride,5-norbornene-2,3-dicarboxylic anhydride, itaconic anhydride, citraconicanhydride, and dodecenylsuccinic anhydride. The amino alkylfunctionality can be, for example, N-(2-hydroxyethyl) iminodiaceticacid, bis(2-hydroxyethyl)amino acetic acid, N-ethyldiethanolamine,4-(2-hydroxyethyl)morpholine, 4-(2-hydroxypropyl) morpholine,N-allyl-2,2′-iminodiethanol, triethanolamine,3-morpholino-1,2-propanediol, 2,6-dimethyl-4-morpholineethanol, andN,N′-diethanolamine.

The chemical bonding of the reactive protein denaturant to the proteincan be covalent or ionic bonding, and the particular type of bondingwill depend on the types of protein and denaturant. For example, soybeanprotein contains about twenty different amino acids, including thosethat contain reactive functional groups such as amino, carboxyl andhydroxyl groups. When the denaturant is maleic anhydride, one of thecarbonyl groups of the maleic anhydride can react with an amino group ofthe protein, with the carbonyl carbon covalently bonding to the aminonitrogen. The bonded maleic anhydride extends from the protein with asecond carbonyl group on the outer end that is capable of furtherreaction as discussed below. Similarly, when the denaturant isacetoacetoxyethyl methacrylate, one of the carbonyl groups of theacetoacetoxyethyl methacrylate can react with an amino group of theprotein, with the carbonyl carbon covalently bonding to the aminonitrogen. The bonded acetoacetoxyethyl methacrylate extends from theprotein with a second carbonyl group on the outer end that is capable offurther reaction.

The chemical bonding of the reactive protein denaturant to the proteinpromotes unfolding of the protein. By way of background, a protein is along strand of amino acids linked together in a specific sequence. Inits usual state, the protein is folded or curled up on itself so thathydrophobic portions of the protein are on the inside of the structureand hydrophilic portions are on the outside. The bonding of thedenaturant to the protein, either alone or in combination with otherfactors, causes the protein to change from its folded structure to asubstantially unfolded structure.

In certain embodiments, the denaturant is hydrophilic and it bonds to ahydrophobic portion of the protein structure, which promotes theunfolding of the protein. Also, in certain embodiments, the denaturantis acidic in character, which promotes the unfolding of the protein.Other factors that could contribute to the unfolding of the proteininclude, for example, heating, adjusting the pH, applying otherchemicals, and the use of certain solvents.

The reaction of the denaturant with the protein can be carried out underany suitable conditions. In some embodiments, the reaction is carriedout in solution, and in other embodiments it is carried out in emulsion.

In other embodiments, the reactive protein denaturant is a polymer thatis functionalized with a reactive moiety. For example, in certainembodiments, the polymer is a graft polymer including the reactivemoiety. The reactive moiety can be any that is capable of chemicallybonding to the protein and promoting unfolding of the protein. Forexample, the reactive moiety can be any of those materials describedabove.

The polymer can be any suitable polymer for including the reactivemoiety in reaction with the protein. It may be a thermoplastic or athermoset polymer, and it may be a homopolymer or a copolymer, dependingon the particular application. For example, in certain embodiments, thepolymer is selected from polyolefins such as polyethylenes,polypropylenes, polybutadienes, polybutenes or polybutylenes. In certainother embodiment, the polymer is selected from polystyrenes or polyvinylethers. Other examples of polymers that may be used include polyesters,polyurethanes, polyamides, polyimides, polysulfones, polyacrylates, andhalogenated polymers.

In another embodiment, the invention provides a method of producing apolymer composite. The method includes providing a polymer in a liquidstate. For example, the polymer can be a hot molten thermoplasticpolymer, or it can be a thermoset polymer which is in a liquid statebefore curing. The method also includes providing a plant material thatincludes protein and carbohydrate, and providing a reactive proteindenaturant that is capable of chemically bonding to the protein andpromoting unfolding of the protein.

In a single-step process, a dispersion is formed of the plant materialand the reactive protein denaturant in a matrix of the liquid polymer,and the reactive protein denaturant and the plant material are reactedto chemically bond them together, using process conditions sufficient tounfold the protein. The reaction of the reactive protein denaturant withthe plant material is included as part of the same process as theforming of the dispersion of plant material and denaturant in thepolymer, rather than a multiple step process in which the reactionoccurs in an initial step and then the dispersion with the polymer isformed in a subsequent step. The polymer can either be in a liquid statebefore the single-step process, or it can be converted from a solid to aliquid state during the process.

Lastly, the polymer is solidified to produce a polymer composite. Forexample, if the polymer in a liquid state is a hot molten thermoplasticpolymer, it can be solidified by cooling it. If the polymer in a liquidstate is a thermoset polymer, it can be solidified by curing it.

In certain embodiments, the reactive protein denaturant bonded to theplant material includes a reactive functional group that reacts with thepolymer. For example, the denaturant may cross-link with the polymer toproduce a more stable composite. As discussed above, denaturants such asmaleic anhydride and acetoacetoxyethyl methacrylate include carbonylgroups that can react with the polymer or with other materials in thecomposite. Other denaturants may have other reactive functional groups.

The polymer used in the composite can be any type of thermoplastic orthermoset polymer suitable for producing a composite. Such polymers arewell-known in the art. For example, the polymer can be a thermoplasticpolymer that is melt-processable between about 130° C. and about 300° C.Some examples of polymers that may be used include polypropylenes,polyethylenes, other polyolefins, polystyrenes, polyalkyl acrylates,chloropolymers such as polyvinyl chlorides, polyesters, polyurethanes,polysulfones, polyamides, polycarbonates, polylactic acids,polyacrylamides, polyetheretherketones, and acrylonitrile butadienestyrenes. Many other types of polymers may also be used; for example,polysuccinates, polyacrylates, and polymers with poly(lactic acid). Thepolymer does not have to be a high molecular weight polymer.

As described above, in certain embodiments the reactive proteindenaturant is a non-polymeric material, and in other embodiments thedenaturant includes a functional material grafted to a polymer. In someembodiments, the polymer portion of the denaturant is the same as thepolymer which forms the matrix of the composite, and in otherembodiments the polymers are different.

In certain embodiments, the reactive protein denaturant is a polymericmaterial, and the denaturant bonded to the plant material provides athermal barrier that protects the plant material from thermal damagewhen it is contacted with the hot molten polymer during production ofthe composite. It is believed that the polymeric portion of thedenaturant may absorb heat and/or shield the plant material from contactwith the hot molten polymer.

Also, in certain embodiments, during the production of the compositeusing a hot molten thermoplastic polymer, the heat from the moltenpolymer promotes the unfolding of the protein. This may provide accessto the reactive functional groups of the protein, which assists thereactive protein denaturant in reacting with and bonding to the protein.

The plant material can be included in the composite in any suitableamount. For example, in some embodiments the amount of the plantmaterial present in the composite is within a range of from about 10 wt% to about 40 wt % depending on the application.

The method of producing a polymer composite can use an suitableequipment, which is well-known in the art. Also, any suitable processingconditions can be used.

In another embodiment, the invention provides a reactive extrusionmethod of producing a polymer composite. The method can be similar tothe single-step process described above, but more particularly theprocess takes place in an extruder. The reactive extrusion methodincludes providing a polymer in a liquid state, providing a plantmaterial that includes protein and carbohydrate, and providing areactive protein denaturant that is capable of chemically bonding to theprotein and promoting unfolding of the protein. In an extruder, adispersion is formed of the plant material and the reactive proteindenaturant in a matrix of the liquid polymer. The reactive proteindenaturant is reacted with the plant material to chemically bond themtogether. The process conditions are sufficient to unfold the protein.The dispersion is extruded into a desired shape. Then the polymer issolidified to produce the polymer composite.

Any suitable extrusion equipment and process conditions can be used toproduce the polymer composite. Any type of suitable extrusion methodscan be used, including direct extrusion, indirect extrusion orhydrostatic extrusion. Most common is direct extrusion with a twin screwor single screw extruder.

In certain embodiments, the temperature of the process in the extruderis controlled. For example, the process may be conducted at atemperature not higher than 360° F., more particularly not higher than350° F., more particularly not higher than 340° F., more particularlynot higher than 330° F., and most particularly not higher than 320° F.Controlling the temperature during the process can result in less odorand less residual color in the product composite.

In certain embodiments, the denaturant used in the process isnonpolymeric (e.g., maleic anhydride). For example, in the extruder insitu process, the process is conducted so that the denaturantpreferentially reacts with the molten polymer (e.g., polypropylene). Forexample, a radical initiator can be used to get them to react together.At the same time, the denaturant reacts with the protein; this resultsin unfolding the entanglement with carbohydrates of the protein, andreacting of the denaturant with the amine groups of the protein.

In certain embodiments, a method of producing a polymer compositeincludes the following. A plant material as described above, for examplesoymeal, and a reactive protein denaturant are dispersed in a moltenpolymer. The soymeal is reacted with the denaturant, and the denaturantis reacted with the polymer. In certain embodiments, this is asingle-step process conducted in an extruder, by a reactive extrusionmethod. For example, the process can include a single-step condensationreaction. This method can produce a product that has long polymeric armsattached to the denaturant and soymeal and practically no short onessuch as those which would be produced by reaction with prepolymers. Theproduct made by this method can be predominantly aliphatic hydrocarbon.

In another embodiment, the invention provides a moldable polymercomposite. The composite comprises: a thermoplastic polymer; a plantmaterial dispersed in a matrix of the polymer, the plant materialincluding carbohydrate and protein that is unfolded; and a reactiveprotein denaturant chemically bonded to the protein and having promotedunfolding of the protein.

The composite can be molded, to produce a molded article, by anysuitable molding process. Such processes and suitable equipment arewell-known in the art. For example, the composite can be injectionmolded, injection blow molded or compression molded into an article.

In certain embodiments, a molded composite product according to theinvention has similar mechanical properties compared to a fullypetroleum derived polymer product. For example, in certain embodiments,the composite product has similar impact strength compared to purepolypropylene. Also, in certain embodiments, the composite product doesnot absorb water on exposure to high humidity.

Polymer composite products may be susceptible to degradation bybacteria. In certain embodiments, the physical properties of a moldedcomposite product according to the invention are not substantiallyaffected by bacterial degradation. In a particular embodiment, thetensile strength of the composite product is not substantially affected.Example 10 hereinbelow presents a study showing no effect on the tensilestrength of the composite by bacterial degradation.

The modified plant materials can be used in many industrialapplications, such as making composites with petroleum based polymer, asfunctional fillers for thermoset resins, as additives in paints andcoatings, and in superabsorbent polymers. It is expected that thematerials can find uses in industries such as transportation, packaging,building and construction, electrical and electronic, furniture andfurnishings, consumer and institutional products, industrial/machinery,adhesives, inks and coatings.

The modified plant material can be used as an anti-caking additive. Itcan function as a crystal growth inhibitor.

A polymer composite produced with the modified plant material can beused to construct a bioreactor for a biorefinery, for example a tubularbioreactor. Because the physical properties of the composite are notsubstantially affected by bacterial degradation, the composite can beuseful as a reactor for the growth of bacteria and other microorganisms.

EXAMPLES Example 1 Soymeal in Polyethylene Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For this example, used only product obtained between 106 μm and75 μm particle size.

Step 2: Protonate SURLYN

Charge 100 g SURLYN 8940 (ethylene/methacrylic acid copolymer) and 1 L1.5 M sulfuric acid into a 1 L reaction kettle fitted with an overheadstirrer (IKA-Werke) (PTFE blade), thermocouple, argon inlet and argonoutlet. Maintain ambient temperature under argon flow for 24 hours.

Rinse product with de-ionized water until washings are pH neutral. Dryin Shel Lab 1410 vacuum oven at 90° C. overnight.

Step 3: Modify Soymeal with SURLYN 8940 (Master Batch)

In a Brabender Type Six Mixer with fitted roller blades, perform thefollowing:

Time Temp Action 0.00 170° F. Add 5 g SURLYN 8940 0.15 170° F. IncreaseSP to 185° F. 0.25 185° F. Increase SP to 200° F. 0.30 200° F. Add 5 gSURLYN 8940 0.45 205° F. Add 10 g soymeal 1.00 215° F. Increase SP to235° F. 1.10 235° F. Add 5 g SURLYN 8940 1.20 251° F. Start addingremaining 35 g soymeal 1.25 251° F. Decrease SP to 215° F. 1.40 240° F.Remove mix from pot

This master batch forms a solid mass. Hammer the mass with a mortar andpestle into pieces smaller than 0.2 inch in diameter.

Step 4: Soymeal/SURLYN Co-extrusion with Polyethylene

Step 4a: Formulation

The following formulations are prepared using both the master batchformulation (from Step 3) and direct addition of 106-75 μm soymeal. TheSURLYN 8940 is the protonated material used in Step 3. The polyethylene(PE) is AT 280 from AT Plastics.

Actual SURLYN Actual MB Actual Neat Actual PE 8940 Formulations (3:1)(grams) Soymeal (grams) (grams) (grams) Control-1 20 Control-2 13.9 6Control-3A 4.5 14 1.51 Control-3B 7.5 10.01 2.5 Control-3C 10.5 5.993.49 Experimental 1 6 14.0 Experimental 2 10 10.0 Experimental 3 14 6.0

Step 4B: Sample Prep

The formulations are dried in a Shel Lab 1410 vacuum oven with Edwardspump set to 140° F. to 160° F. and maximum vacuum (35 mm Hg) overnightbefore extruding.

Step 4C: Extrusion

For each of these 20 grams formulations above, the materials aremanually inserted into a twin screw mini extruder (Thermo Haake MINILAB)at a temperature of 240° F. with screws rotating at 50-60 RPM. Theformula is fed into the screws, manually packing with a brass rod. Cutthe extruded strand into pieces smaller than 2 inches in length.

Step 5: Compression Molding of PE Composite Material (20 Grams Batches)Step 5A: Setup

With a Phi press set both bottom and top platens to a temperature of250° F. Soak two 0.5 inch aluminum cover plates until the temperatureequilibrates (about 5 minutes). The aluminum plates are covered inKAPTON polyimide film (DuPont).

Step 5B: Melting Resin

After bringing the plates to temperature, the resin is placed on thecenter of the bottom platen. The plates are separated with a brass shim.Close the press in order to bring the top and bottom plates in contactwith the heated press platens. Soak the resin at temperature in thepress for 3 min (timed with a stopwatch). Periodically adjust the platesto maintain contact with the platens.

Step 5C: Pressing

After the 3 min, the pressure is increased to 15 tonnes for 30 sec(timed with a stopwatch).

Step 5D: Cooling

Finally, the plates are removed and put into a water cooled cold press(Wabash Hydraulic Press model 30-1221) for at least 3 minutes untilcool. The result is an approximately 6-inch diameter film, at about 5mils in thickness.

Step 6: Tensile Testing of Composite Films

Samples are cut into 0.5 inch strips and tested for tensile propertiesusing a 5564 model Instron universal test machine, equipped with Merlinsoftware. The parameters are as follows:

-   Load cell: 224.8 lb-   Sample width: 0.5 in-   Sample gauge length: 1.0 in-   Sample thickness: average of 3 measurements in test area and    manually entered-   Test Rate: 0.5 in/min-   Data interval: 100 ms

The following tensile results were obtained for the PE films:

Thickness Extension at Max. Load Strain at Stress at Extension at Youngs(in) Break (in) (lbf) Max (%) Max (psi) Max (in) Modulus (psi) C1 Ave0.0050 1.50 2.72 89.55 1103.73 0.90 14308.62 Std 0.0003 0.27 0.13 20.0689.43 0.20 2132.50 C2 Ave 0.0060 0.83 3.81 82.01 1276.19 0.82 18496.60Std 0.0002 0.15 0.09 14.79 41.27 0.15 1186.46 C3A Ave 0.0070 0.11 2.768.93 800.67 0.19 19843.16 Std 0.0006 0.02 0.33 1.73 68.23 0.02 660.08C3B Ave 0.0080 0.01 3.21 6.10 856.68 0.06 26872.88 Std 0.0002 0.02 0.361.58 100.30 0.02 2532.64 C3C Ave 0.0070 0.05 3.41 4.56 936.14 0.0537409.54 Std 0.0004 0.01 0.34 0.95 113.99 0.01 4379.72 E1 Ave 0.00600.19 3.16 15.12 1102.50 0.15 22618.12 Std 0.0003 0.04 0.34 2.77 87.970.03 1494.51 E2 Ave 0.0060 0.09 3.50 8.24 1173.14 0.08 34304.86 Std0.0008 0.02 0.42 1.64 90.45 0.02 4808.47 E3 Ave 0.0070 0.06 4.31 5.901314.63 0.06 47128.60 Std 0.0003 0.01 0.25 1.01 82.56 0.01 5785.89

Example 2 Soymeal in Polypropylene Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For this example, used only product obtained between 106 μm and75 μm particle size.

Step 2: Modify Soymeal with Polypropylene-Graft-Maleic Anhydride (MasterBatch)

In a Brabender Type Six Mixer with fitted roller blades, perform thefollowing:

Time Temp Action 0.00 193° F. Add 10 g PP 0.15 193° F. Add 10 g soymeal0.20 193° F. Add 15 g soymeal 0.35 198° F. Increase SP to 210° F. 0.40206° F. Add 5 g soymeal 0.45 224° F. Increase SP to 235° F. 0.55 235° F.Increase SP to 245° F. 1.05 245° F. Increase SP to 265° F. 1.35 265° F.Remove material from potStep 3: Soymeal/SURLYN Co-extrusion with Polypropylene (20 g Batches)

Step 3A: Formulation

The following formulations are prepared using both the master batchformulation (from Step 2 above) and direct addition of 106-75 μmsoymeal. The polypropylene-graft-maleic anhydride is from Sigma-Aldrich.The polypropylene is from Total Petrochemicals (melt index of 35 g/10min).

Actual Neat Actual Actual MB Soymeal Actual PE SURLYN Formulations (3:1)(grams) (grams) (grams) 8940 (grams) PP_Control-1 20 PP_Control-2 14 6PP_Control-3α 2 17.3 0.74 PP_Control-3β 4.49 14 1.56 PP_Control-3γ 7.59.99 2.6 PP_Experimental 2.72 17.3 PP_Experimental 6.03 14.05PP_Experimental 10.02 10 PP_Control-3δ 2 17.7 0.31 PP_Control-3ε 2 17.90.2 PP_Control-3ζ 2.05 18

Step 3B: Sample Preparation

The formulations are dried in a Shel Lab 1410 vacuum oven with Edwardspump set to 130° F. to 140° F. and maximum vacuum (35 mm Hg) overnightbefore extruding.

Step 3C: Extrusion

Manually insert 20 grams of PP formulation into a twin screw miniextruder (Thermo Haake MINILAB) at a temperature of 320° F. with screwsrotating at 60-70 RPM. The extruded strand is cut into pieces smallerthan 2 inches in length.

Step 4: Compression Molding of PP Composite Material (20 g Batches) Step4A: Setup

With a Phi press set both bottom and top platens to a temperature of325° F. Soak two 0.5 inch aluminum cover plates until the temperatureequilibrates (about 5 minutes). The aluminum plates are covered inKAPTON polyimide film (DuPont).

Step 4B: Melting Resin

After bringing the plates to temperature, 2 grams of resin are placed onthe center of the bottom platen. The plates are separated with a brassshim. Close the press in order to bring the top and bottom plates incontact with the heated press platens. Soak the resin at the settemperature for 2 min (timed with a stopwatch). Periodically adjust theplates to maintain contact with the hot platens.

Step 4C: Pressing

After the 3 min, the pressure is increased to 15 tonnes for 30 sec(timed with a stopwatch).

Step 4D: Cooling

Finally, the plates are removed and put into a water cooled cold press(Wabash Hydraulic Press model 30-1221) for at least 3 minutes untilcool. The result is an approximately 6-inch diameter film, at about 5mils in thickness.

Step 5: Tensile Testing of PP Composite Films (20 g Batches)

Samples are cut into 0.5 inch strips and tested for tensile propertiesusing a 5564 model Instron universal test machine, equipped with Merlinsoftware. The parameters are the same as in Example 1.

The following tensile results were obtained for the PE films:

Thickness Extension at Max. Load Strain at Stress at Extension at Youngs(in) Break (in) (lbf) Max (%) Max (psi) Max (in) Modulus (psi) PP_C1 Ave0.0060 3.30 11.13 14.41 3948.47 0.14 134868.00 Std 0.0001 2.81 0.38 1.39136.70 0.01 7822.67 PP_C2 Ave 0.0040 0.09 7.00 8.62 3948.50 0.09155728.85 Std 0.0003 0.02 0.66 1.85 270.75 0.02 6001.86 PP_C3α Ave0.0060 0.11 10.39 9.23 3696.24 0.09 143121.09 Std 0.0008 0.04 0.63 2.06389.29 0.02 15327.54 PP_C3β Ave 0.0060 0.07 10.04 6.84 3651.54 0.07146897.84 Std 0.0005 0.02 1.00 1.22 275.66 0.01 10030.79 PP_C3γ Ave0.0030 0.04 9.85 3.69 3169.27 0.04 170112.44 Std 0.0010 0.01 1.15 0.77520.95 0.01 27915.22 PP_E1 Ave 0.0060 0.12 10.20 8.62 3649.83 0.09143313.72 Std 0.0006 0.03 0.90 1.50 420.36 0.01 16199.12 PP_E2 Ave0.0060 0.06 9.73 5.93 3294.20 0.06 146169.35 Std 0.0008 0.01 1.01 1.10403.68 0.01 23937.72 PP_E3 Ave 0.0060 0.03 9.31 3.39 3071.60 0.03169571.88 Std 0.0009 0.01 1.31 0.73 446.40 0.01 14468.89 PP_C3δ Ave0.0060 0.10 10.16 8.39 3475.02 0.08 143141.01 Std 0.0006 0.03 1.28 2.15545.08 0.02 11136.30 PP_C3ε Ave 0.0060 0.12 10.53 9.35 3603.45 0.09144388.33 Std 0.0011 0.05 1.14 2.54 526.13 0.03 12914.38 PP_C3ζ Ave0.0050 0.16 9.05 9.81 3474.02 0.10 151729.30 Std 0.00 0.09 0.40 2.35807.25 0.02 34272.77

Example 3 Soymeal in Polypropylene (Direct Addition) Scale-up Step 1:Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For experimentation, use only product obtained between 106 μm and75 μm particle size.

Step 2: Formulation of Composite—10% Soymeal, 1.5% PP-g-MA in PP

The formulation is the same as sample PP_C3δ in Example 2, but scalebatch up to 20 pounds.

Combine soymeal (907.5 g (2 lbs)) and PP-graft-MA (136.08 g (0.3 lbs))(Aldrich), and dry materials in a Fisher Scientific ISOTEMP gravity ovenat 60° C.

Perform a regression study on the powder mixture (above) and the PPresin (8028.7 g (17.7 lbs)) in two separate feeder hoppers. The feedersare turned on for 1 minute and the output is collected in a bucket andweighed. Three measurements are performed at each rate setting. Theaverage output weights (grams/min) were obtained with the following flowrate settings.

Feeder 1 Rate Setting PP Average  25 43.9 41.8 41.3 42.33  50 88.2 83.492.3 87.97  75 127.8 136 129.5 131.10 100 177.2 174.9 176.9 176.33Feeder 2 Rate Setting Soy + PP-g-MA Average  25 8.9 7.9 8.8 8.53  5016.6 17.5 15.9 16.67 100 34 34.5 32.7 33.73 200 70.9 66.2 69.8 68.97 300102.9 101.8 102.9 102.53

Using Excel, the results are plotted (not shown) to get an equation forthe flow rates of each feeder. The total flow rates are calculated usingthe equations for each feeder. For this formulation, the total flow is11.5% powder mixture (PP-g-MA and soymeal) and 88.5% PP resin.

11.5% Soy + PP-g-MA Total Flowrate (g/min) Soy + PP-g-MA PP Feeder 1Feeder 2 150 17 133 71 49 200 23 177 95 66 250 29 221 119 83 300 35 266143 100 350 40 310 166 116 400 46 354 190 133

Step 3: Extrude Material

The sample is extruded in a Berstorff 3016987 extruder, fixed with aspaghetti strand die at 200 g/min. To achieve this rate, Feeder 1 is setto 95 and feeder 2 is set to 66. Allow the extruder to warm up to theset temperatures before turning the feeders on. The extruder speed isset to 78 and the extruder voltage reads 78 volts. When the soy andresin flow, the torque (extruder amps) reads 28 to 29.5 A and the melttemperature is 316.3° F.

The extruder settings are as follows:

Zone W0 B2 B3 B4 B5 B6 B7 B8 H9 Initial Values at the Beginning of theRun Current 91 295 301 313 309 310 305 313 315 Tem- pera- ture Set 85295 300 310 310 310 310 310 315 Tem- pera- ture Final Values at the Endof the Run Current 92 301 290 305 304 305 303 304 312 Tem- pera- tureSet 85 290 290 305 305 305 305 305 310 Tem- pera- ture

The extruded material is pulled through a 6 ft Berlyn Tap Water Bath,and the strand is pelletized with a Berlyn HV-1 pelletizer. The pelletsare approximately 0.1 inch in length. The pelletized composite materialis put into an oven overnight at 60° C. to dry.

Step 4: Injection Mold Extruded PP Composite Scale-up (201b Batch)

Dry material from Step 3 is processed by injection molding in aCincinnati injection molder set to make 2.5 inch dog bone tensile barsand 0.125 inch plaques.

Step 5: Tensile Testing of PP Composite Dog Bones

Samples are molded into dog bone shapes 0.5×0.125×2.5 inches. They aretested for tensile properties using a 4505 model Instron universal testmachine, equipped with Series 9 software. The parameters are as follows:

-   Load cell: 2248 lb-   Sample width: 0.5 in-   Sample gauge length: 2.5 in-   Sample thickness: 0.125 in-   Test Rate: 0.5 in/min-   Data interval: 25 pts/sec-   Data reduction: 0.3 min

The tensile testing results are as follows:

Max. Strain Stress Extension Youngs Load at Max at Max at Max Modulus(lbf) (%) (psi) (in) (psi) Control Ave 253.20 12.38 4052.00 0.31170920.20 Std 3.30 1.66 53.00 0.04 15458.14 Sample 1 Ave 253.70 8.214060.00 0.21 182170.72 (315° F.) Std 5.40 1.18 87.00 0.03 18090.27Sample 2 Ave 254.30 8.30 4069.00 0.21 183389.77 (335° F.) Std 0.90 0.6815.00 0.02 5791.26 Sample 3 Ave 239.20 8.47 3827.00 1.21 163714.64 (355°F.) Std 1.00 0.53 17.00 0.01 9103.55 Sample 4 Ave 225.30 10.35 3605.000.26 158712.58 (380° F.) Std 0.80 0.63 13.00 0.02 5167.30 Sample 5 Ave215.00 12.58 3440.00 0.31 151295.98 (420° F.)

Example 4 Soymeal in Polystyrene (Emulsion) Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For experimentation, use only product obtained between 106 μm and75 μm particle size.

Step 2: Pre-Emulsion

In a 400 mL beaker (Beaker 1), combine 100 mL de-ionized water and 10.01g sodium dodecylsulfate. Place the beaker in a crystallization dish andfill the dish with ice. Stir the beaker contents with an overheadstirrer fitted with a dispersion blade.

In a separate 400 mL beaker (Beaker 2), combine 2.62 g AIBN and 140.7 gstyrene monomer. Cover with a watch glass and place on a magnetic stirplate until the AIBN has been dissolved completely. Transfer thesolution to a 250 mL separatory funnel.

Add the contents of the separatory funnel dropwise to Beaker 1 whilestill under dispersion. Using a powder addition funnel, slowly add 10.87g (106-75 μm) soymeal to Beaker 1. During addition of soymeal, sonicatethe contents of Beaker 1 three times at 54 watts for 1 minute each time(Misonix SONICATOR 3000). Transfer contents of Beaker 1 to a 250 mLseparatory funnel.

Step 3: Polymerization

In a 500 mL reaction kettle fitted with an overhead stirrer,thermocouple, and argon inlet and outlet, heat 100 mL de-ionized waterto 60° C. under stirring and argon flow. Remove argon outlet port andinsert neck of separatory funnel into kettle. Add contents of separatoryfunnel to the kettle dropwise. Once all material has been charged,maintain kettle at 60° C. for 4 hours. Allow product to cool understirring.

Transfer product to glass bottle, add 0.20 g biocide, and stir contents.

Example 5

Modification of Soymeal with AAEM (Solution)

Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For experimentation, use only product obtained between 106 μm and75 μm particle size.

Step 2: Modification with AAEM

In a 1 L reaction kettle fitted with an overhead stirrer, an argoninlet, a thermocouple, and a condenser (with argon outlet), charge thefollowing: 80.0 g (106-75 μm) soymeal, 720.0 g DMAc, 8.0 g AAEM, and 0.1g TEA. Start argon and condenser flow and turn on overhead stirrer.React at 80° C. for 12 hours. Allow product to stir while cooling toroom temperature.

Step 3: Product Isolation

Transfer kettle contents to a 1 L centrifuge bottle and centrifuge (IECDPR-6000 Centrifuge) for 15 minutes at 3,000 RPM. Decant the supernatantand replace with 500 g distilled water. Repeat previous step threeadditional times. Dry the resulting solid in a Shel Lab 1410 vacuum ovenat 70° C. overnight.

Remove product from oven and grind on a Glen Mills MICRO HAMMER CUTTERMILL IV through a 0.2 mm screen. Collect powder in a glass bottle.

Example 6

Modification of Soymeal with AAEM (Emulsion)

Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For experimentation, use only product obtained between 106 μm and75 μm particle size.

Step 2: Pre-Emulsion

In a 400 mL beaker (Beaker 1), combine 100 mL de-ionized water and 9.99g sodium dodecylsulfate. Place the beaker in a crystallization dish andfill the dish with ice. Stir the beaker contents with an overheadstirrer fitted with a dispersion blade.

In a separate 400 mL beaker (Beaker 2), combine 2.60 g AIBN, 100 mL MMA,and 40.04 g AAEM. Cover with a watch glass and place on a magnetic stirplate until the AIBN has been dissolved completely. Transfer thesolution to a 250 mL separatory funnel.

Add the contents of the separatory funnel dropwise to Beaker 1 whilestill under dispersion. Using a powder addition funnel, slowly add 10.47g (106-75 μm) soymeal to Beaker 1. During addition of soymeal, sonicatethe contents of Beaker 1 three times at 54 watts for 1 minute each time(Misonix SONICATOR 3000). Transfer contents of Beaker 1 to a 250 mLseparatory funnel.

Step 3: Polymerization

In a 500 mL reaction kettle fitted with an overhead stirrer,thermocouple, and argon inlet and outlet, heat 100 mL de-ionized waterto 60° C. under stirring and argon flow. Remove argon outlet port andinsert neck of separatory funnel into kettle. Add contents of separatoryfunnel to the kettle dropwise. Once all material has been charged,maintain kettle at 60° C. for 4 hours. Allow product to cool understirring.

Transfer product to glass bottle, add 0.20 g biocide, and stir contents.

Example 7

Modification of Soymeal with MMA, BA and Soy Acrylate

Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For experimentation, use only product obtained between 106 μm and75 μm particle size.

Step 2: Pre-Emulsion

In a 400 mL beaker (Beaker 1), combine 100 mL de-ionized water and 10.03g sodium dodecylsulfate. Place the beaker in a crystallization dish andfill the dish with ice. Stir the beaker contents with an overheadstirrer fitted with a dispersion blade.

In a separate 400 mL beaker (Beaker 2), combine 2.62 g AIBN, 100.01 gMMA, 29.98 g BA and 10.04 g soy acrylate. Cover with a watch glass andplace on a magnetic stir plate until the AIBN has been dissolvedcompletely. Transfer the solution to a 250 mL separatory funnel.

Add the contents of the separatory funnel dropwise to Beaker 1 whilestill under dispersion. Using a powder addition funnel, slowly add 10.86g (106-75 μm) soymeal to Beaker 1. During addition of soymeal, sonicatethe contents of Beaker 1 three times at 54 watts for 1 minute each time(Misonix SONICATOR 3000). Transfer contents of Beaker 1 to a 250 mLseparatory funnel.

Step 3: Polymerization

In a 500 mL reaction kettle fitted with an overhead stirrer,thermocouple, and argon inlet and outlet, heat 100 mL de-ionized waterto 60° C. under stirring and argon flow. Remove argon outlet port andinsert neck of separatory funnel into kettle. Add contents of separatoryfunnel to the kettle dropwise. Once all material has been charged,maintain kettle at 60° C. for 4 hours. Allow product to cool understirring.

Transfer product to glass bottle, add 0.20 g biocide, and stir contents.

Example 8

Modification of Soymeal with MMA, BA and Soy Acrylate

Step 1: Process Soymeal

Grind soymeal (ADM, HI-PRO) using a hammer mill (MIKRO BANTAM fromHoskawa Micron Powder Systems) through a 0.010 HB slot screen (254microns). Sift (Gilson SS-8R Sieve Shaker) the ground product using 300μm, 106 μm, 75 μm and 45 μm sieves (USA Standard Testing Sieve; ASTME-11Spec). For experimentation, use only product obtained between 106 μm and75 μm particle size.

Step 2: Pre-Emulsion

In a 400 mL beaker (Beaker 1), combine 100 mL de-ionized water and 10.01g sodium dodecylsulfate. Place the beaker in a crystallization dish andfill the dish with ice. Stir the beaker contents with an overheadstirrer fitted with a dispersion blade.

In a separate 400 mL beaker (Beaker 2), combine 2.61 g AIBN, 105.00 gMMA, 20.03 g BA and 14.94 g soy acrylate. Cover with a watch glass andplace on a magnetic stir plate until the AIBN has been dissolvedcompletely. Transfer the solution to a 250 mL separatory funnel.

Add the contents of the separatory funnel dropwise to Beaker 1 whilestill under dispersion. Using a powder addition funnel, slowly add 10.58g (106-75 μm) soymeal to Beaker 1. During addition of soymeal, sonicatethe contents of Beaker 1 three times at 54 watts for 1 minute each time(Misonix SONICATOR 3000). Transfer contents of Beaker 1 to a 250 mLseparatory funnel.

Step 3: Polymerization

In a 500 mL reaction kettle fitted with an overhead stirrer,thermocouple, and argon inlet and outlet, heat 100 mL de-ionized waterto 60° C. under stirring and argon flow. Remove argon outlet port andinsert neck of separatory funnel into kettle. Add contents of separatoryfunnel to the kettle dropwise. Once all material has been charged,maintain kettle at 60° C. for 4 hours. Allow product to cool understirring.

Transfer product to glass bottle, add 0.20 g biocide, and stir contents.

Example 9 Part 1: Procedure for Making Unsaturated Polyester ResinSample A:

In a 1000 mL reaction kettle fitted with a thermocouple, overheadstirrer, Dean Stark trap and inter gas inlet, charged Cargill X-0210(bio-based polyol) (395 grams), propylene glycol (228 grams) andisophthalic acid (93 grams). The contents in the reactor were heated to215 deg C. and cook till the acid value of the reaction mixture lessthan 15 mg KOH/g of sample. The reaction mixture was cooled to 110 degC. and added maleic anhydride (235 grams) and hydroquinone (100 partsper million). The contents were heated to 210 deg C. and continued tillthe acid value is around 35 mg of KOH/g of sample. The product obtainedis cooled. To 65 parts of the product thus obtained, 31.5 parts ofstyrene and 3.5 parts of acetoacetoxy ethyl methacrylate were mixed andstored.

Sample B:

In a 250 mL reaction kettle fitted with a thermocouple, overheadstirrer, Dean Stark trap and inert gas inlet, chargedcis-5-Norbornene-endo-2,3-dicarboxylic anhydride (50 grams),bis(2-hydroxyethyl)amino acetic acid (104 grams), dibutyl tin oxide(0.15 grams), toluene (70 mL) and dimethyl acetamide (120 mL). Thecontents were heated to reflux and continued the reaction till the acidvalue is around 250 mg of KOH/g of sample. The solvent was removed underpressure at 60 deg C. to obtain a prepolymer. To 50 gram of thisprepolymer, added 55 grams of Cargill X-0210 (bio-based polyol) and0.007 grams of hydroquinone. The contents were heated to 210 deg C. andcontinued till the acid value is around 20 mg of KOH/g of sample. Theproduct obtained is cooled. To 65 parts of the product thus obtained,31.5 parts of styrene and 3.5 parts of acetoacetoxy ethyl methacrylatewere mixed and stored.

Sample C:

In a 250 mL reaction kettle fitted with a thermocouple, overheadstirrer, Dean Stark trap and inert gas inlet, chargedcis-5-Norbornene-endo-2,3-dicarboxylic anhydride (50 grams),bis(2-hydroxyethyl)amino acetic acid (104 grams), dibutyl tin oxide(0.15 grams), toluene (70 mL) and dimethyl acetamide (120 mL). Thecontents were heated to reflux and continued the reaction till the acidvalue is around 250 mg of KOH/g of sample. The solvent was removed underpressure at 60 deg C. to obtain a prepolymer. To 30 gram of thisprepolymer, added 55 grams of Cargill X-0210 (bio-based polyol), 4.32grams of maleic anhydride and 0.007 grams of hydroquinone. The contentswere heated to 210 deg C. and continued till the acid value is around 20mg of KOH/g of sample. The product obtained is cooled. To 65 parts ofthe product thus obtained, 31.5 parts of styrene and 3.5 parts ofacetoacetoxy ethyl methacrylate were mixed and stored.

Performance of unsaturated polyester resin soybean meal composites.

Sample D:

A fiber glass composite made from sample A and soybean meal had thefollowing properties.

Property Value Viscosity-(Brookfield LVT 880 cps #3 @30 RPM) NonVolatile % 69 Acid Number-On solution 14 (mg/g) Specific Gravity(lbs./gallon) 8.87 SPI Gel Time (minutes) 3.53 SPI Cure (minutes) 3.05from Gel (Total 6.58) SPI Peak Exotherm (Degrees 175 C.) Dielectric CureAnalysis- 11.93 Minimum Flow Time (seconds) Dielectric Cure Analysis-Gel21.36 Time (seconds) Dielectric Cure Analysis- 53.06 Cure Time (seconds)Specific Gravity 1.78 Shrink (Hot Tool to Cold 2.76 Part) mil./inchShrink (Cold Tool to Cold 1.26 Part) Paste Viscosity before 8000 cps at90 F. Thickener Viscosity in 1 Day (millions 3 CPS) Stored 95 F.Viscosity in 4 Days (millions 11 CPS) Stored 95 F. Molded Panel at20-25% Yes Glass

Example 10 Effect of Bacteria on Tensile Strength of Composite

This study demonstrated that the tensile strength of a soy-polymercomposite material was not affected when incubated with two specifictypes of bacteria in liquid culture for four weeks. This does not meanthat the composite material is not susceptible to degradation by alltypes of bacteria, but this does suggest that bacteria may not affectthe physical properties of this material. Additional studies would benecessary to further prove that this material is not affected bybacterial degradation. These could include experiments such as exposingthe strips to a consortium (mixture) of bacteria in liquid media andcovering the strips with soil to allow soil microbes to act upon them.

To evaluate the effect of microbial degradation on a soy polymercomposite material according to the invention, a test method wasdeveloped that initially measured the effect of microbial growth on thetensile strength of this soy-polymer material. The objective of thisstudy was to determine the effect of bacterial growth under controlledconditions on the tensile strength of the soy polymer composite.

The approach used for this study was to incubate soy-polymer compositestrips with bacteria in selected media with controls. A stock culturewas obtained from the American Type Culture Collection (ATCC).Pseudomonas putida ATCC 17472, a common environmental soil bacterium,was selected for use on this study. This organism has the ability tosurvive on a minimal amount of nutrients and can degrade many types ofmaterials. The test design used four sets of flasks described inTable 1. One flask of each condition was prepared for each anticipatedtime point (0, 1, 2, 3 and 4 weeks). Soy strips were surfacedecontaminated with alcohol using a saturated cloth while being heldwith sterile forceps before placing them into the flasks and incubatingat 26° C.

TABLE 1 Experimental Design Sample ID Medium Organism Soy Strips Soy 1Minimal P. putida + Soy 2 Minimal None + Soy 3 Nutrient Broth P. putida− Soy 4 Minimal P. putida − Note: Mimimal media is also referred to asBasal Inorganic Minimal Medium, Recipe B (BIMB)

After incubation with the bacteria, test strips were removed, samplessurface decontaminated by wiping with alcohol, and the tensile strengthof the test strips was measured by AMPE to determine if the bacterialgrowth was affecting the physical properties of the material.

Methods used in the conduct of the testing are described in the sectionsbelow. The methods include preparation of the test organism from thefreeze-dried stock provided by ATCC, inoculation of test flasks,investigation of a contamination source, and demonstration that thebacteria were associated with the test strips and could metabolize soymeal.

Organism Preparation

The freeze-dried stock of P. putida was rehydrated according to ATCCinstructions by adding nutrient broth (NB) to the lyophilized culture inthe glass vial. The stock was incubated for 48 hours at 26° C. andstreaked onto a nutrient agar (NA) plate for isolation to determinepurity and create a working stock plate that was used for thisexperiment. The working stock plate containing the culture was incubateduntil the colonies were of a countable size. Colonies from this platewere used to inoculate nutrient broth as a starter culture for the testset up. The starter culture was incubated until turbid and then used toinoculate flasks for the experiments described in Table 1.

Sample Collection and Enumeration

Starting at zero hours, and once a week for four weeks, the soy stripswere removed from the appropriate flasks, surface decontaminated withalcohol, and given to the AMPE group to measure tensile strength.Samples of the liquid from the flasks were added to NA plates to becultured for enumeration to determine the concentration of P. putida inthe flask at that time point. Serial dilutions of the culture were madeusing PBS and enumerated after incubating the plates until the coloniesreached a distinguishable size. Plates were counted and the colonyforming units per mL (cfu/mL) were calculated.

Tests to Determine Source of Contamination

Bacterial contamination was observed in all samples after the first weekof incubation. Samples that were not inoculated with P. putida were alsoturbid and contained bacterial colonies when streaked for isolation onagar plates. Various methods were used to characterize the bacterialcontaminant and determine its origin. Gram stain microscopy wasperformed to determine the Gram reaction, cell characteristics, and todetermine if more than one type of bacteria was present. The media usedto grow and manipulate the bacteria was tested for the presence ofviable organisms by streaking the liquids onto nutrient agar plates andincluded NB, BIMB, and PBS. To confirm the contamination was not on theNA plates, some were placed into an incubator without opening them. Theenvironment inside the biosafety cabinet was tested by placing open NAplates within the cabinet during operation. The pipette tips were testedfor the presence of viable organisms by placing a tip from the box beingused in a tube containing NB. The P. putida working stock plate wasre-streaked on to NA plates for isolation to confirm that only P. putidawas present as a pure culture. Flasks containing NB were autoclaved todemonstrate the autoclave effectively sterilized media in the flasks. Asoy strip was checked for contaminating organisms by wiping the stripwith alcohol and placing it into a tube with nutrient broth. All sampleswere incubated at 37° C. and observed for bacterial growth.

Demonstration of Bacteria Attached to Soy Strips

A test was done to determine if bacteria were associated with thesurface of the soy-polymer test strip. A strip from the Soy 1 test groupat week three was removed from the flask, rinsed with sterile water andthen placed onto a NA plate and incubated at 37° C. until visible growthwas seen.

Demonstration of P. putida Growth on Soy Meal

A test was set up to demonstrate that the P. putida could utilize soymeal to grow. Bactosoytone, an enzymatic digest of soybean meal that isan additive for bacterial media, was added to PBS or BIMB and filtersterilized. This was then split into two sterile tubes. One wasinoculated with P. putida from the NA working stock culture plate usinga sterile loop. The tubes were incubated at 37° C. with shakingovernight and evaluated visually for turbid growth.

Results:

After the first week of growth, contamination was observed in allsamples #1-4. The bacterial concentrations (cfu/mL) for both P. putidaand the contaminating organism are reported in Table 2. The pure stockstreak of P. putida showed 3-5 mm cream colored, smooth, round, concavecolony morphology. The alternate morphology of the contaminatingbacteria was pinpoint, whitish colonies. The alternate colony morphologywas observed in all samples, including the ones with no P. putida addedand with BIMB, which theoretically has no carbon source for vegetativegrowth. Both the contaminating bacteria and the P. putida were Gramnegative rods. The source of contamination was not identified.

TABLE 2 Estimated Concentration of Bacteria in Samples Flasks Counts inCFU/mL over Time Sample 0 Week 1 Week 2 Week 3 Week 4 Soy 13.40E+06 >1E+3 1.30E+08 7.20E+08 9.70E+07 Soy 2 Not counted Not counted1.70E+06 1.60E+06 1.20E+06 Soy 3 3.40E+06 >1E+5 4.30E+06 1.10E+079.50E+06 Soy 4 3.30E+06 >1E+3 2.80E+07 1.40E+07 2.90E+07 Note: Allcounts are estimates due to presence of contaminating bacteria

The sample (Soy 1) that was spiked with P. putida and contained soystrips had higher concentrations of bacteria compared to the othersamples including the control flasks (Soy 2) that were not spiked withP. putida. This may indicate that the P. putida was utilizing the stripsto grow. In the other cultures, Soy 3 and Soy 4, the bacterialconcentration remained relatively constant. This was anticipated in Soy4 since there was no carbon source. In Soy 3, where P. putida wasinoculated into nutrient broth, some level of growth was expected.

The soy test strips were shown to have bacteria binding to the surfaceas demonstrated by the thick bacterial growth seen all around the teststrip.

The P. putida bacteria used for this experiment were able to metabolizesoy protein. The tubes containing Bactosoytone that were inoculated withthe P. putida were very turbid indicating bacterial growth while theones not inoculated remained clear.

DISCUSSION AND CONCLUSIONS

This study intended to determine the effect of P. putida bacterialgrowth on the tensile strength of a soy-based polymer. Following fourweeks of growth, all test flasks contained high concentrations ofbacterial growth, including control flasks anticipated to have nogrowth. The test strips did not show any change in tensile strength inany of the test conditions compared to the initial values at time zeroeven though bacteria were found in the un-inoculated control flask. Thisindicates that the tensile strength of the soy-polymer composite did notchange in the presence of the two specific types of bacteria, P. putidaand the contaminant bacteria, over the course of four weeks. The resultsdo not mean that the strips are not susceptible to degradation by alltypes of bacteria; however P. putida was chosen because of its abilityto degrade many types of complex materials and should be a goodpreliminary indicator that the material may not be affected by bacterialgrowth. Additional testing should be performed to further characterizethe biodegradability of the soy-polymer composite. These tests couldinclude incubating the test strips with a mixture of bacterial strainsand assessing degradation in a matrix containing a consortium ofbacteria and fungi. A mixture of bacteria may use multiple mechanisms todegrade the strips and may act synergistically to enhance the effect.Burying the test strips in soil would allow naturally occurring, butnon-culturable bacteria to degrade the test strips.

APPENDIX A: MEDIA FORMULATION Basal Inorganic Minimal Media, Recipe B(BIMB, or Minimal Media)

-   -   KH₂PO₄—0.2 g/L    -   K₂HPO₄—0.8 g/L    -   MgSO₄.7H₂O—0.5 g/L    -   (NH₄)₂SO₄—1 g/L    -   FeSO₄.7H₂O—0.01 g/L    -   CaSO₄—0.03 g/L

Example 11 Maleic Anhydride Denaturant

Mix ingredients into a paste after charging into PARR autoclave reactor:hammermilled soymeal (30 grams), maleic anhydride (30 grams), anddistilled water (175 grams). Close lid and pressurize to 1280 PSI. Setheater to 40 C and mix at 150 RPM. After about an hour the temperatureovershot to 50 C and the pressure increased to 1475 PSI. Then afteranother hour, the temperature equilibrated to 40 C and the pressuredecreased to 1411 PSI. The next day the reactor was cooled and theproduct was isolated by filtration. The product particle size isprovided below:

-   -   Vol. Weighted Mean D[4,3]: 34.248 um    -   Surface Weighted Mean D[3,2]: 15.920 um

Example 12 Maleated Soybean Oil Denaturant

Combine the following: polypropylene (24 grams), soymeal (6 grams),maleated soybean oil (1.2 grams). These ingredients were mixed togetherby roll-mill overnight. The following day it was extruded in the ThermoHaake mini extruder (settings 375 F/45 RPM).

Example 13 Maleated Methyl Soyate Denaturant

Combine the following: polypropylene (24 grams), soymeal (6 grams),maleated methyl soyate (1.2 grams). These ingredients were mixedtogether by roll-mill overnight. The following day it was extruded inthe Thermo Haake mini extruder (settings 375 F/45 RPM).

Example 14 Maleated Partially Hydrogenated Soybean Oil Denaturant

Combine the following: polypropylene (24 grams), soymeal (6 grams),maleated partially hydrogenated soybean oil (2.4 grams). Theseingredients were mixed together by roll-mill overnight. The followingday it was extruded in the Thermo Haake mini extruder (settings 375°F./45 RPM).

Example 15

Composites with Less Odor and Residual Color

The soybean meal used in the above four examples is Arsoy™ which has aprotein content of 32% (AOAC) and a carbohydrate content of 57%. Thissoybean meal was compared with a soybean meal having a protein contentof 49% and a carbohydrate content of 34% (hereinafter referred to as the“Control”). It was surprisingly found that when composites are formedfrom lower protein soymeal the resultant final product had less odor andresidual color.

The different soymeal samples were inserted into sealed vials andexposed to temperatures of 250° F., 315° F., 355° F. and 400° F. Thesamples were submitted for GC-MS. It was noted that color change above355° F. was more pronounced in the Control soymeal sample having ahigher protein content. Headspace gas analysis of the sealed vials wasconducted. Furan and pyrazine derivatives are major components in soydegradation which causes malodors (e.g., burnt meat, musty, caramel orwoody odors). The Control soymeal sample having a higher protein contentevolved significantly greater amounts of furan, methyl pyrazine, anddimethyl disulfide gases than the Arsoy™ soymean sample having the lowerprotein content.

In further experimentation, it was found that lower protein content andoptimal particle size and processing temperature can control degradationof soymeal. Samples of different particle size and protein content werecompounded. These were then injection molded at different temperatures.The extruded formulations were as follows: 10% Control (49 microns); 27%Control (49 microns); 10% Arsoy™ (76 microns), 27% Arsoy™ (76 microns),10% Arsoy™ (<15 microns), 27% Arsoy™ (<15 microns), 10% Arsoy™ (<15microns) with 3% Shulman additive, 27% Arsoy™ (<15 microns), with 3%Shulman additive, 10% soy hulls (134 microns) containing 9% protein and86% carbohydrates.

The samples were exposed to temperatures of 315° F., 355° F. and 400° F.Color evaluation showed that the Arsoy™ produced parts with lessbrowning compared with the Control and with hulls. The additive furtherreduced browning. Odor qualitative analysis showed that the Arsoy™product had less odor than the Control product.

1. A method of producing a polymer composite comprising: providing apolymer in a liquid state; providing a plant material that includesprotein and carbohydrate, the plant material having a particle size lessthan 50 microns; providing a reactive protein denaturant; forming adispersion of the plant material and the reactive protein denaturant ina matrix of the liquid polymer, reacting to chemically bond the plantmaterial to the reactive protein denaturant, and reacting to chemicallybond the reactive protein denaturant to the polymer; and solidifying thepolymer to produce the polymer composite.
 2. The method of claim 1wherein the plant material has a particle size less than 20 microns. 3.The method of claim 1 wherein the plant material has a protein contentless than 45%.
 4. The method of claim 1 wherein the plant materialcomprises a soybean material.
 5. The method of claim 1 wherein thereactive protein denaturant is a material selected from ethylenicallyunsaturated anhydrides, ethylenically unsaturated carboxylic acids,ethylenically unsaturated carboxylic acid esters, ethylenicallyunsaturated amines and imines, ethylenically unsaturated diketonates,and/or derivatives of these materials.
 6. A reactive extrusion method ofproducing a polymer composite, the method comprising: providing apolymer in a liquid state; providing a plant material that includesprotein and carbohydrate, the plant material having a particle size lessthan 50 microns; providing a reactive protein denaturant; in asingle-step process in an extruder, forming a dispersion of the plantmaterial and the reactive protein denaturant in a matrix of the liquidpolymer, reacting to chemically bond the plant material to the reactiveprotein denaturant, reacting to chemically bond the reactive proteindenaturant to the polymer, and extruding the dispersion; and solidifyingthe polymer to produce the polymer composite.
 7. The method of claim 6wherein the plant material has a particle size less than 20 microns. 8.The method of claim 6 wherein the plant material has a protein contentless than 45%.
 9. The method of claim 8 wherein the plant material has aprotein content less than 35%.
 10. The method of claim 6 wherein theprocess in the extruder is conducted at a temperature not higher than360° F.
 11. The method of claim 6 wherein the plant material comprises asoybean material.
 12. The method of claim 6 wherein the reactive proteindenaturant bonded to the plant material includes a reactive functionalgroup which reacts with the polymer.
 13. The method of claim 6 whereinthe reactive protein denaturant is a non-polymeric material.
 14. Themethod of claim 13 wherein the reactive protein denaturant is a materialselected from ethylenically unsaturated anhydrides, ethylenicallyunsaturated carboxylic acids, ethylenically unsaturated carboxylic acidesters, ethylenically unsaturated amines and imines, ethylenicallyunsaturated diketonates, and/or derivatives of these materials.
 15. Themethod of claim 6 wherein the reactive protein denaturant includes afunctional material grafted to a second polymer.
 16. A moldable polymercomposite comprising: a thermoplastic polymer; and a plant materialdispersed in a matrix of the polymer, the plant material includingcarbohydrate and protein that is unfolded, the plant material having aparticle size less than 50 microns; and a reactive protein denaturantchemically bonded to the protein and having promoted unfolding of theprotein.
 17. The polymer composite of claim 16 wherein the plantmaterial has a particle size less than 20 microns.
 18. The polymercomposite of claim 16 wherein the plant material has a protein contentless than 45%.
 19. The polymer composite of claim 18 wherein the plantmaterial has a protein content less than 35%.
 20. The polymer compositeof claim 16 wherein the plant material comprises a soybean material.